Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants

Degradation of milk components through various enzymatic activities associated with the contamination of dairy products by Pseudomonas spp. can reduce the shelf life of processed milk. Reliable methods for differentiating among Pseudomonas spp. strains are necessary to identify and eliminate specific sources of bacterial contamination from dairy processing systems. To that end, we assessed the genetic diversity and dairy product spoilage potentials among a total of 338 Pseudomonas spp. isolates from raw and pasteurized milk and from environmental samples collected from four dairy processing plants. The majority of isolates were identified as P. fluorescens and P. putida by API 20 NE. A total of 42 different ribotype patterns were identified among a subset of 81 isolates. The presence of many different ribotypes within this collection indicates high genetic diversity among the isolates and suggests multiple origins of contamination within the processing plant and in dairy products. The extracellular enzyme activity patterns among Pseudomonas isolates appeared to be associated with ribotypes. Isolates with the same ribotype frequently had the same extracellular protease, lecithinase, and lipase activities. For example, isolates grouped in ribotype 55-S-6 had the highest extracellular protease activity, while those in ribotypes 50-S-8 and 72-S-3 had the highest extracellular lipase activities. We conclude that ribotyping provides a reliable method for differentiating Pseudomonas strains with dairy food spoilage potential.     

Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples

Food products, such as milk and meat products including cheese, milk powder, fermented milk, sausage, etc. are susceptible to the contamination by pathogenic and deteriorative bacteria. These bacteria include Listeria monocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and Pseudomonas fluorescens, etc. Traditional methods for the detection of these microorganisms are laborious and time consuming. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and PCR primers for the detection of aforementioned microorganisms. By using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected. When the system was used for the inspection of milk or meat homogenate containing 10(0) target cells per milliliter or gram of the sample, all these bacterial species could be identified after an 8h pre-enrichment step. The system consisting of a multiplex PCR step followed by macroarray allowed us to detect multiple target bacterial species simultaneously without the use of agarose gel electrophoresis. Compared to the commonly used multiplex PCR method, this approach has the additional advantage of detecting more bacterial strains because some bacterial strains generate PCR products with the same molecular sizes which can be differentiated by macroarray but not by electrophoresis.     

Phenotypic and genotypic characterization of non-starter lactic acid bacteria in mature cheddar cheese.

Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of Lactobacillus-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II) Lactobacillus spp. Phenotypically, the isolates comprised 96.4% L. paracasei, 2.1% L. plantarum, 0.3% L. curvatus, 0.3% L. brevis, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic Lactobacillus spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the L. paracasei complex.     

Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewes' milk cheese

AIMS: To assess the intraspecific genetic diversity of lactococci and enterococci isolated from 24-h, 1- and 2-month-old home-made Pecorino Sardo ewes' milk cheese. METHODS AND RESULTS: Two molecular techniques, plasmid profiling and pulsed-field gel electrophoresis, were used in order to type the isolates at strain level. The present study revealed that the lactococcal and enterococcal microbial populations of home-made Pecorino Sardo cheese were complex, not only 24 h after manufacture, but also after 1 and 2 months of ripening. The genetic diversity at subspecies level ranged from 58 to 80% during the three periods examined. The study also showed that the strains that dominated in the first stage of ripening were not necessarily predominant in the later periods. A high number of strains isolated at 24 h were still present in the mature cheese, but many of the genotypes were only found in the cheese after 1 or 2 months. CONCLUSIONS: The results showed a high intraspecific genetic diversity in the natural microbial population colonizing home-made Pecorino Sardo cheese. Two molecular techniques are necessary for a thorough and precise typing at strain level in order to better distinguish between closely related isolates and between isolates that probably belong to the same clonal lineage. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic complexity observed in the present study is of particular relevance in the preservation of the natural microflora of traditional Protected Designation of Origin raw milk cheeses, as well as in the selection of new starter strains for the dairy industry.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.     

Surface microbial consortia from Livarot, a French smear-ripened cheese

The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR (rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG)(5)-PCR, and when appropriate by 16S rDNA sequencing and SDS-PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly assigned to the genera Arthrobacter , Brevibacterium , Corynebacterium , and Staphylococcus . New taxa related to the genera Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed a high level of diversity and mainly included members of the genera Alcaligenes , Hafnia , Proteus , Pseudomonas , and Psychrobacter . Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gram-negative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of their positive versus negative role should be objectively examined.     

Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

AIMS: Pseudomonas spp. are considered the most important milk spoilage organisms. Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp. in milk. METHODS AND RESULTS: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas. Twenty different Pseudomonas spp. and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested. All tested Pseudomonas spp. yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction. The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species. The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h. Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products. CONCLUSIONS: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp. in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.     

Genotypic and phenotypic heterogeneity among lactococci isolated from traditional Pecorino Sardo cheese

Twenty-nine Lactococcus lactis isolates from one traditional 24 h-old Pecorino Sardo cheese were characterized phenotypically, technologically and genotypically in order to assess the biodiversity within this wild microbial population. Two DNA-based techniques, plasmid profiling and PFGE, were used for the genetic typing of the isolates. All 29 isolates were characterized at strain level and eight different genotypes were recognized. In addition, by combining the results from plasmid profile analysis and PFGE, it was possible to identify closely related isolates probably belonging to the same clonal lineage. The dominant biotype was identified in the 24 h-old cheese, as were the strains believed to act as starters for the curd. Atypical lactococci, able to grow in 6.5% NaCl, were isolated. The results suggest that wild bacterial populations should be preserved in order to protect the traditional raw milk cheeses, and to select new starter strains for the dairy industry.     

Source of enterococci in a farmhouse raw-milk cheese

Enterococci are widely distributed in raw-milk cheeses and are generally thought to positively affect flavor development. Their natural habitats are the human and animal intestinal tracts, but they are also found in soil, on plants, and in the intestines of insects and birds. The source of enterococci in raw-milk cheese is unknown. In the present study, an epidemiological approach with pulsed-field gel electrophoresis (PFGE) was used to type 646 Enterococcus strains which were isolated from a Cheddar-type cheese, the milk it was made from, the feces of cows and humans associated with the cheese-making unit, and the environment, including the milking equipment, the water used on the farm, and the cows' teats. Nine different PFGE patterns, three of Enterococcus casseliflavus, five of Enterococcus faecalis, and one of Enterococcus durans, were found. The same three clones, one of E. faecalis and two of E. casseliflavus, dominated almost all of the milk, cheese, and human fecal samples. The two E. casseliflavus clones were also found in the bulk tank and the milking machine even after chlorination, suggesting that a niche where enterococci could grow was present and that contamination with enterococci begins with the milking equipment. It is likely but unproven that the enterococci present in the human feces are due to consumption of the cheese. Cow feces were not considered the source of enterococci in the cheese, as Enterococcus faecium and Streptococcus bovis, which largely dominated the cows' intestinal tracts, were not found in either the milk or the cheese.     

Contamination of milk by enterococci and coliforms from bovine faeces

AIM: To determine the contribution of enterococci and coliforms from bovine faeces and teats to contamination of raw milk. Methods: Putative enterococci (n = 301) and coliforms (n = 365) were isolated from bovine faeces (n = 20), cows' teats (n = 20), the raw milk (n = 1) and the milking environment (n = 4) on one farm. The clonal relationships of each bacterial group were investigated using Pulsed-Field Gel Electrophoresis of genomic macrorestriction fragments. Representatives of the different clusters of enterococci were identified by molecular techniques including rep-PCR, SDS protein profiling, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), phenylalanyl-tRNA synthase (pheS) sequence analysis and/or 16S rDNA gene sequencing. Coliforms were identified by API 20E strips. RESULTS: The majority of the bovine faecal enterococcal isolates were identified as a potential new species of Aerococcus (100 isolates); E. faecium (28 isolates), and Aerococcus viridans (28 isolates) were also found. All coliform isolates from the bovine faeces were identified as Escherichia coli. The coliforms present in the milk were Hafnia alvei, Serratia liquefaciens, Yersinia enterocolitica and Enterobacter amnigenus. No E. coli, Enterococcus or Aerococcus from the bovine faeces were found in the milk. A single clone of H. alvei was found in the water, the milking equipment and the milk, suggesting that the water was the source of the organism in the milk. No vancomycin-resistant aerococci or enterococci were found while most of the isolates tested showed the presence of at least one virulence gene. The milk-sock retained strains that adhered to particulate faecal material. Coliforms were present at approx. 2 orders of magnitude greater than enterococci in the bovine faeces. CONCLUSIONS: The results imply that bovine faeces are not an important source of contamination of raw milk with enterococci or coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm those of two previous studies (Gelsomino et al. 2001, Int J Food Microbiol71, 177-188 and Kagkli et al. 2007, Int J Food Microbiol114, 243-251) on two other farms. The three studies show that contamination of milk by enterococci, lactobacilli and coliforms of bovine faecal origin is extremely low. The results also suggest that where raw milk is implicated in food infection, other factors in addition to faecal contamination must be involved.     

Characterization of Listeria monocytogenes isolated from production lines of fresh and cold-smoked fish

AIMS: The aims of this study were to characterize strains of Listeria monocytogenes isolated from cold-smoking fish plants to establish possible routes of contamination through the processing chain. METHODS AND RESULTS: Listeria monocytogenes from fresh fish suppliers, raw materials, factory sites and finished products isolated in Portugal (162 isolates) and England (28 isolates) were characterized by serotyping, phage typing, tetracycline, cadmium and arsenic resistance, and plasmid profiling. On the basis of serotyping and phage typing, the isolates were categorized into eight groups. Although cultures within some of the groups could be further differentiated on the basis of plasmid profiling and cadmium and arsenite typing, consideration of all typing data predominantly clustered together isolates from a single location. L. monocytogenes strains: from fresh salmon suppliers were not found in the processing lines; from fresh salmon from different locations differed; and from the water where salmon trout were farmed differed from those isolated from the fish samples. SIGNIFICANCE AND IMPACT OF THE STUDY: No clear source or route of contamination in the cold-smoked processing chain could be established; however, these results highlight the complexity in tracking this bacterium through food chains.     

Determining the source of Bacillus cereus and Bacillus licheniformis isolated from raw milk, pasteurized milk and yoghurt

Aims:  Strain-specific detection of Bacillus cereus and Bacillus licheniformi s in raw and pasteurized milk, and yoghurt during processing.
Methods and Results:  Randomly selected isolates of Bacillus spp. were subjected to PCR analysis, where single primer targeting to the repetitive sequence Box elements was used to fingerprint the species. The isolates were separated into six different fingerprint patterns. The results show that isolates clustered together at about the 57% similarity level with two main groups at the 82% and 83% similarity levels, respectively. Contamination with identical strains both of B. cereus and B . licheniformis in raw and pasteurized milk was found as well as contaminated with different strains (in the case of raw milk and yoghurt/pasteurized milk and yoghurt). Several BOX types traced in processed milk samples were not discovered in the original raw milk.
Conclusions:  BOX-PCR fingerprinting is useful for characterizing Bacillus populations in a dairy environment. It can be used to confirm environmental contamination, eventually clonal transfer of Bacillus strains during the technological processing of milk.
Significance and Impact of the Study:  Despite the limited number of strains analysed, the two Bacillus species yielded adequately detectable banding profiles, permitting differentiation of bacteria at the strain level and showing their diversity throughout dairy processing.     

Distribution and phylogeny of hexachlorocyclohexane-degrading bacteria in soils from Spain

Hexachlorocyclohexane (HCH)-degrading bacteria are believed to mediate natural attenuation of HCH contamination and have potential for active bioremediation processes. This study addressed the very limited understanding of the distribution, diversity and substrate specificity of such bacteria from 13 soil samples, varying in levels of HCH contamination, from four sites in Spain. Hexachlorocyclohexane removal occurred in 16 of 36 enrichment cultures. Hexachlorocyclohexane-degrading populations were clearly associated with HCH-contaminated soils, and populations growing on the delta-HCH isomer were only found in soil contaminated with delta-HCH. beta-Hexachlorocyclohexane was persistent in enrichment cultures, and there was no evidence for populations growing on beta-HCH. From alpha- and gamma-HCH enrichment cultures, nine HCH-degrading isolates were obtained, which were all Sphingomonas spp. Attempts to isolate organisms from delta-HCH enrichment cultures failed. None of the isolates grew on HCH as a sole organic substrate in pure culture. All isolates degraded alpha- and gamma-HCH, and most degraded beta-HCH. delta-Hexachlorocyclohexane inhibited growth of most isolates, but could be degraded by cell suspensions of at least four strains. Denaturing gradient gel electrophoresis indicated that the isolates represented predominant populations in the enrichment cultures, but additional predominant populations, including some Pseudomonas spp., could not be isolated.     

Lantibiotics biosynthesis genes and bacteriocinogenic activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. isolated from raw milk and cheese

Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by 16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or 48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods.     

Phenotypic and Genotypic Characterization of Non-Starter Lactic Acid Bacteria in Mature Cheddar Cheese

Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of Lactobacillus-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II) Lactobacillus spp. Phenotypically, the isolates comprised 96.4% L. paracasei, 2.1% L. plantarum, 0.3% L. curvatus, 0.3% L. brevis, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic Lactobacillus spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the L. paracasei complex.     

Surveillance and genotyping of Enterobacter sakazakii suggest its potential transmission from milk powder into imitation recombined soft cheese

Aims:  To examine the presence of Enterobacter sakazakii in milk and milk-related products produced/distributed under Egyptian conditions and to probe possible transmission routes of the pathogen during the preparation of dairy products.
Methods and Results:  One hundred and thirty-seven samples of milk and milk-related products were randomly collected from Egyptian markets and examined for the presence of Ent. sakazakii . The pathogen could be detected only in skimmed milk powder (SMP) and its related product, imitation recombined soft (IRS) cheese. Enterobacter sakazakii isolates recovered from these products were phenotypically similar and sensitive to all antibiotics examined in this study. They also showed indistinguishable banding patterns when subjected to macro-restriction profiling using pulsed-field gel electrophoresis (mrp-PFGE). One Ent. sakazakii isolate was inoculated into SMP that was used in the preparation of IRS cheese using two cheese making procedures. The pathogen could survive for up to 1 month in the IRS cheese prepared by either procedure.
Conclusions:  The simultaneous presence of Ent. sakazakii in SMP and IRS cheese samples collected within the same local market besides the phenotypic and genotypic similarities of isolates recovered from these samples suggested the possibility of Ent. sakazakii being transmitted from SMP into IRS cheese. This hypothesis was supported by the observation that the pathogen could survive in the IRS cheese prepared from contaminated SMP.
Significance and Impact of the Study:  The study highlights SMP and IRS cheese as potential transmission vehicles of Ent. sakazakii . It also raises concern on the microbiological safety of IRS cheese prepared from SMP.     

Source of Enterococci in a Farmhouse Raw-Milk Cheese

Enterococci are widely distributed in raw-milk cheeses and are generally thought to positively affect flavor development. Their natural habitats are the human and animal intestinal tracts, but they are also found in soil, on plants, and in the intestines of insects and birds. The source of enterococci in raw-milk cheese is unknown. In the present study, an epidemiological approach with pulsed-field gel electrophoresis (PFGE) was used to type 646 Enterococcus strains which were isolated from a Cheddar-type cheese, the milk it was made from, the feces of cows and humans associated with the cheese-making unit, and the environment, including the milking equipment, the water used on the farm, and the cows' teats. Nine different PFGE patterns, three of Enterococcus casseliflavus, five of Enterococcus faecalis, and one of Enterococcus durans, were found. The same three clones, one of E. faecalis and two of E. casseliflavus, dominated almost all of the milk, cheese, and human fecal samples. The two E. casseliflavus clones were also found in the bulk tank and the milking machine even after chlorination, suggesting that a niche where enterococci could grow was present and that contamination with enterococci begins with the milking equipment. It is likely but unproven that the enterococci present in the human feces are due to consumption of the cheese. Cow feces were not considered the source of enterococci in the cheese, as Enterococcus faecium and Streptococcus bovis, which largely dominated the cows' intestinal tracts, were not found in either the milk or the cheese.     

Biodiversity of lactic acid bacteria in Romanian dairy products

Traditionally fermented dairy products are still a very important part of the daily food in Romania, especially for people living in the countryside. To study the biodiversity of lactic acid bacterium strains of these products, 110 samples (raw and fermented milk, sour cream, and cheese) were collected from farm houses, monasteries, and local markets throughout Romania. Lactic acid bacteria (LAB) were isolated using six different cultivation conditions. All 599 isolates were tested for their Gram reaction, catalase activity, and morphology. A rep-PCR fingerprinting technique with the (GTG)5 primer and, in some cases SDS-PAGE of total cell proteins and 16S rRNA gene sequencing were used to cluster and/or identify the LAB. The biodiversity of the isolated strains was correlated with the type of product and/or technology applied. The most frequent LAB found in Romanian raw milk and fermented dairy products were Lactococcus lactis, Leuconostoc spp., and Enterococcus spp. Among the latter, a new species E. saccharominimus was found.     

Cytotoxic Bacillus spp. belonging to the B. cereus and B. subtilis groups in Norwegian surface waters

AIMS: To investigate the presence and numbers of Bacillus spp. spores in surface waters and examine isolates belonging to the B. cereus and B. subtilis groups for cytotoxicity, and to discuss the presence of cytotoxic Bacillus spp. in surface water as hazard identification in a risk assessment approach in the food industry. METHODS AND RESULTS: Samples from eight different rivers with variable degree of faecal pollution, and two drinking water sources, were heat shocked and examined for the presence of Bacillus spp. spores using membrane filtration followed by cultivation on bovine blood agar plates. Bacillus spp. was present in all samples. The numbers varied from 15 to 1400 CFU 100 ml(-1). Pure cultures of 86 Bacillus spp. isolates representing all sampling sites were characterized using colony morphology, atmospheric requirements, spore and sporangium morphology, and API 50 CHB and API 20E. Bacillus spp. representing the B. cereus and B. subtilis groups were isolated from all samples. Twenty-one isolates belonging to the B. cereus and B. subtilis groups, representing eight samples, were screened for cytotoxicity. Nine strains of B. cereus and five strains belonging to the B. subtilis group were cytotoxic. CONCLUSIONS: The presence of cytotoxic Bacillus spp. in surface water represents a possible source for food contamination. Filtration and chlorination of surface water, the most common drinking water treatment in Norway, do not remove Bacillus spores efficiently. This was confirmed by isolation of spores from tap water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of food with water containing low numbers of Bacillus spores implies a risk for bacterial growth in foods. Consequently, high numbers of Bacillus spp. may occur after growth in some products. High numbers of cytotoxic Bacillus spp. in foods may represent a risk for food poisoning.     

Effect of fusaric acid and phytoanticipins on growth of rhizobacteria and Fusarium oxysporum

Suppression of soilborne diseases by biocontrol agents involves complex interactions among biocontrol agents and the pathogen and between these microorganisms and the plant. In general, these interactions are not well characterized. In this work, we studied (i) the diversity among strains of fluorescent Pseudomonas spp., Bacillus spp., and Paenibacillus sp. for their sensitivity to fusaric acid (FAc) and phytoanticipins from different host plants, (ii) the diversity of pathogenic and nonpathogenic Fusarium oxysporum isolates for their sensitivity to phytoanticipins, and (iii) the influence of FAc on the production of pyoverdine by fluorescent Pseudomonas spp. tolerant to this compound. There was a great diversity in the response of the bacterial strains to FAc; however, as a group, Bacillus spp. and Paenibacillus macerans were much more sensitive to FAc than Pseudomonas spp. FAc also affected production of pyoverdine by FAc-tolerant Pseudomonas spp. strains. Phytoanticipins differed in their effects on microbial growth, and sensitivity to a phytoanticipin varied among bacterial and fungal strains. Biochanin A did not affect growth of bacteria, but coumarin inhibited growth of Pseudomonas spp. strains and had no effect on Bacillus circulans and P. macerans. Conversely, tomatine inhibited growth of B. circulans and P. macerans. Biochanin A and tomatine inhibited growth of three pathogenic isolates of F. oxysporum but increased growth of three nonpathogenic F. oxysporum isolates. Coumarin inhibited growth of all pathogenic and nonpathogenic F. oxysporum isolates. These results are indicative of the complex interactions that can occur among plants, pathogens, and biological control agents in the rhizosphere and on the root surface. Also, these results may help to explain the low efficacy of some combinations of biocontrol agents, as well as the inconsistency in achieving disease suppression under field conditions.